A rational basis for the development of antisense therapies of diseases whose etiology is characterized by, or associated with, specific DNA or RNA sequences has been recognized for a number of years. The basic principle is straightforward enough. The ultimate therapeutic agent, whether directly administered or generated in situ, is an oligomer which will be complementary to a DNA or RNA needed for the progress of the disease. By specifically binding to this target DNA or RNA, the ordinary function of these materials is interdicted.
The logic behind this approach is readily seen in visualizing the administration of, for example, an oligomer having a base sequence complementary to that of an mRNA which encodes a protein necessary for the progress of the disease. By hybridizing specifically to this RNA, the synthesis of the protein will be interrupted. However, it is also possible to bind even double-stranded DNA using an appropriate oligomer capable of effecting the formation of a specific triple helix by inserting the administered oligomer into the major groove of the double-helical DNA. The elucidation of the sequences which form the targets for the therapeutics is, of course, a problem which is specific to each target condition or disease. While the general principles are well understood and established, there remains a good deal of preliminary sequence information required for the design of a particular oligomeric probe.
One feature of the oligomeric probes which is, however, common to most targets is the structuring of the backbone of the administered oligomer so that it is resistant to endogenous nucleases and is stable in vivo, but also retains its ability to hybridize to the target DNA or RNA. (Agarwal, K. L. et al., Nucleic Acids Res (1979) 6:3009; Agarwal, S. et al., Proc Natl Acad Sci USA (1988) 85:7079.) As nucleases attack the phosphodiester linkage, a number of modified oligonucleotides have been constructed which contain alternate linkages. Among these have been methylphosphonates (wherein one of the phosphorous-linked oxygens has been replaced by methyl) phosphorothioates (wherein sulphur replaces one of these oxygens) and various amidates (wherein NH.sub.2 or an organic amine derivative, such as morpholidates or piperazidates, replace an oxygen). These substitutions confer enhanced stability, for the most part, but suffer from the drawback that they result in a chiral phosphorous in the linkage, thus leading to the formation of 2.sup.n diastereomers where n is the number of modified diester linkages in the oligomer. The presence of these multiple diastereomers considerably weakens the capability to hybridize to target sequences. Some of these substitutions also retain the ability to support a negative charge; the presence of charged groups decreases the ability of the compounds to penetrate cell membranes. (Miller, P. S. et al., Biochemistry (1981) 20:1874; Marcus-Secura, C. J. et al., Nucleic Acids Res (1987) 15:5749; Jayaraman, K. et al., Proc Natl Acad Sci USA (1981) 78:1537.) There are numerous other disadvantages associated with these modified linkages, depending on the precise nature of the linkage.
It has also been suggested to use carbonate diesters; however, these are highly unstable, and the carbonate diester link does not maintain a tetrahedral configuration exhibited by the phosphorous in the phosphodiester. Similarly, carbamate linkages, while achiral, confer trigonal symmetry and it has been shown that poly dT having this linkage does not hybridize strongly with poly dA (Coull, J. M., et al., Tet Lett (1987) 28:745; Stirchak, E. P., et al., J Org Chem (1987) 52:4202.
Of relevance to the present invention are modified phosphodiester linkages involving sulfur, rather than oxygen as a linking substituent. See, for example, Cosstick, R., et al., Tet Lett (1989) 30:4693-4696, which describes oligonucleotides containing 3'-thiothymidine.
The general approach to constructing oligomers useful in antisense therapy has been reviewed, for example, by van der Krol, A. R., et al., Biotechniques (1988) 6:958-976, and by Stein, C. A. et al., Cancer Res (1988) 48:2659-2668.
The present invention provides an oligomeric linkage which is isosteric to the phosphodiester found in the native molecule, but which is resistant to nuclease digestion, and which is stable under physiological conditions, and which can be neutral so as to enhance cell permeation. Furthermore, the linkages can be achiral and thus do not lead to the problem of multiple diastereomers in the resulting compounds.